Is a Kazark sequence needed in a upper primer? - (Dec/05/2006 )
If u have plan to amplify some gene, then I would advise to add the Kozak to the genes, but if u plan to use plasmids (without kozak) as they they have been supplied by a company, I wouldnt go thru the whole process of adding the kozak.
that;s perhaps less work. But doesn't it affect expression levels??
Has anyone any information on what the expression levels are like without kozak sequence, especially when doing protein overexpression studies??
CDS is "coding sequence" of your transcript sequence. There's the 5'UTR, CDS and 3'UTR with a polyA.
If the plasmid you bought is "ready-to-use" for expression, it should have Kozak consensus sequence (probably explained in the info sheet?). Sometimes, people like to use consensus sequence intrinsic to the gene itself instead of the strict cgccacc[atg]. You can check NCBI, look at where the CDS starts and compare the 5'UTR (around 10 nts from ATG?) with that of your construct's.
E.g. for actin (gamma) (Accession # NM_001614), it's ccggtcgca[atg]g, where there's a purine (A and G) at -3 (relative to [atg], in which "a" is considered +1) and a "G" is at position +4 which has been cited to be important for ribosome recognition of the start codon.
Can't remember where I got this info. But definitely from google search.
Most important is to see if the construct you got is in cloning vector (need to construct into expression vector) or expression vector ("ready-to-use" type).
can u explain this part please?
here we dont add kozak sequence each time , its a case by case issue, but really i dont know how it works without kozak and sometimes the expression level is even better than with kozak???
Can't seem to figure out how to insert image from C drive (it prompts URL only). Anyway, you can refer to http://www.stratagene.com/manuals/211170.pdf and www.pkclab.org/PKC/vector/pEGFPN1.pdf and look for the start codon. There'll be Kozak sequence around ATG (either short or longer one), then the tag and then the limited RE option for inserting the DNA in.
Hope this helps.
thankx but one last question
in the map u gave me *i will attach it* there is CMV promotor then MCS *i put my insert here* then kozak sequence then EGFP right?
so i dont have to put kozak again in front of my insert ?
You'll have to place Kozak before your insert if you place it within MCS.
NOTE: I've checked the vector properly and it seems that there's a stop codon in GFP sequence. In order to clone it as N-term tagged fusion protein, it's limited to using BsrGI with Not I or Xba I (or BsrG I alone). Also have to ensure it's in correct frame. Haha... prob like this makes me really want to add tag by means of PCR-tagging. More hassle-free.